The clotting process follows a series of autocatalytic reactions eventually forming fibrin, the insoluble protein network which forms the essential portion of the blood clot. The blood clot is first formed from fibrin, which subsequently entangles other blood components such as platelets, white cells, and red cells to form an aggregate. This aggregation of blood components is referred to as a thrombus and the formation, development, or presence of a thrombus is referred to as thrombosis.
Fibrin is formed from fibrinogen by the proteolytic action of thrombin, an enzyme derived from prothrombin, during the normal clotting of blood. When vessels or body cells are damaged, a prothrombin activator is released that converts the prothrombin into thrombin. The thrombin, in the presence of several accessory factors, converts soluble fibrinogen into insoluble fibrin. For example, the rupture of a blood vessel can create a roughened surface to which platelets adhere and partially plug the break. This initiates the development of a prothrombin activator. The prothrombin activator converts prothrombin to thrombin. The thrombin can then convert the fibrinogen to fibrin, the insoluble protein network forming the blood clot. After the function of the clot has been fulfilled, fibrin is normally digested into soluble products.
Plasmin is the active portion of the thrombolytic, or clot-lysing, system and has a high specificity for fibrin. Plasminogen, present in the blood, is converted to plasmin in a reaction that is catalyzed by plasminogen activators such as urinary plasminogen activator (u-PA).
The high molecular weight form of human urinary activator has one kringle domain, or region of sequence homology, with a relatively low fibrin-binding affinity. Pennica et al., Nature (London), 301, 214-221 (1983). Effectively, the human high molecular weight urinary activator, urokinase (UK1), displays no specific affinity to fibrin or fibrinogen.
The native, or naturally occurring, human high molecular weight UK1 is a serine protease that is synthesized in the kidney and excreted into the urine. Two major molecular forms of UK1 have been isolated from human tissues and characterized; a high molecular weight form with a molecular weight of approximately 54,000 daltons, and a low molecular weight form with an approximate molecular weight of 31,000 daltons. The high molecular weight UK1 is considered to be the major native form found in urine and the low molecular weight form of UK1 is considered to be an enzymatically degraded form of the high molecular weight UK1. Both forms have been found to contain two chains linked by a disulfide bond. Hiroyuki et al., J. Biol. Chem., 258, 8014-8019 (1983). The NH.sub.2 -terminal, or A chain, has a single kringle domain that shows extensive homology with the plasminogen kringles, whereas the active center of the enzyme is located in the COOH-terminal, or B chain. Robbins et al., Biochemistry, 25, 3603-3611 (1986).
Nonnative forms of urokinase are collectively described herein as UK2. The high molecular weight urokinase, UK1, can be cleaved between Lysine (Lys) and Isoleucine (Ile), which are located in positions 158 and 159, respectively, as conventionally determined from the NH.sub.2 terminus of the urokinase molecule, to form a two-chain urokinase (UK2.sub.1). The two chains are held together by a single disulfide bond which is located between Cysteine (Cys), in position 148, and Cys, in position 279. This form, UK2.sub.1, is more active toward chromogenic substrates than the high molecular weight form, UK1; however, it also displays increased plasminogen binding. Lijnen et al., J. Biol. Chem., 261, 1253-1258 (1986); Collen et al., J. Biol. Chem., 261, 1259-1266 (1986). A smaller version, UK22, can be formed by cleaving the high molecular weight form of urokinase after Lys, in position 135.
In another version, UK2.sub.3, the molecule begins at Leucine (Leu), in position 144 as numbered in the high molecular weight UK1, and is missing the two amino acids Phenylalanine (Phe) and Lys, in positions 157 and 158, respectively. Similarly, the two chains are held together by a single disulfide bond between Lys and Ile, which are located in positions 158 and 159, respectively. The two chains include a short A chain, having the 13 residue peptide from positions 144 to 156, and a long B chain beginning at position 159, having the COOH-terminal at about position 411 and containing the active center of the enzyme. The 13 residue peptide is referred to herein as the remnant A peptide and has the amino acid sequence Leu-Lys-Phe-Glu-Cys-Gly-Glu Lys-Thr-Leu-Arg-Pro-Arg. The UK2.sub.3 form of urokinase is available commercially and is sold by Abbott Laboratories under the registered trademark "ABBOKINASE".
The UK2 forms of urokinase retain the single kringle domain found in the much larger A chain of high molecular weight UK1 as well as the active center that is located in the COOH terminal, or B chain. Similarly, the UK2 forms of urokinase have no specific affinity to fibrin or fibrinogen.
Current clinical therapies for dissolution of blood clots that occur in myocardial infarction, deep vein thrombosis and pulmonary embolism, often involve the use of UK2 to activate the fibrinolytic system in blood. This systemic activation can cause degradation of fibrinogen and lead to a decrease in circulating plasminogen, as well as other clotting factors. Verstraete, Fibrinolysis, 185-200, CRC Press, Boca Raton, FL (1980). Plasminogen is converted to the active enzyme plasmin. When plasmin circulates freely in the blood it promotes systemic activation, thus, degrading a number of proteins, including fibrinogen. The systemic activation of plasminogen is due to the specific binding affinity of urokinase for plasminogen, wherever found, and the lack of specificity for fibrin that is found at the site of the blood clot. Verstraete, Fibrinolysis, 185-200, CRC Press, Boca Raton, FL (1980). An overt systemic fibrinolytic state and very low fibrinogen levels can occasionally lead to major bleeding. Collen et al., Thrombolysis, 74, 838-842 (1986).
It is desirable, therefore, to incorporate synthetic peptides having specific clot binding properties into the u-PA type of urokinase in order to provide fewer systemic complications and reduce the likelihood of major bleeding.
Prior art attempts have been made to impart clot binding properties to urokinase derivatives. The catalytic carboxyl-terminal domain of residues of UK was combined with the amino-terminal plasmin kringle region recovered from reduced plasmin. A mixture of the two components was allowed to oxidize and the hybrid was isolated. Robbins et al., Biochemistry, 25, 3603-3611 (1986).
Sumi et al., Journal of Biological Chemistry, 258, 8014-8019 (1983), reported the mild reduction and carboxymethylation of high molecular weight urokinase (UK1) with 2-mercaptoethanol to generate two chains, a functionally active heavy chain and a light chain. This process reduced only one connecting disulfide bridge.
An E. coli expressed recombinant high molecular weight single-chain urokinase was expressed and refolded using a glutathione catalyzed system. Winkler and Blaber, Biochemistry, 25, 4041-4045 (1986). This reduction-reoxidation procedure was slow, requiring up to 48 hours in the presence of guanidine, and resulted in a low yield.
Maksimenko et al., Thrombosis Research, 38, 277-288 (1985), generated a heparin-urokinase derivative by carbodiimide promoted coupling of heparin to non-reduced low molecular weight urokinase.
Maksimenko et al., Thrombosis Research, 38, 89-295 (1985), generated another urokinase derivative by attachment of fibrinogen through a spacer of an aliphatic diamine to carbodiimide activated low molecular weight two-chain urokinase.